Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

Reduced Na+ Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB

The melibiose permease of Salmonella enterica serovar Typhimurium (MelB_St) catalyzes symport of melibiose with Na⁺, Li⁺, or H⁺. Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na⁺-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V_max) for melibiose transport in Escherichia coli DW2 and greatly decrease Na⁺ affinity, indicating that intracellular release of Na⁺ is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop_4-5 and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V_max with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.     

Alternating Hemiplegia of Childhood mutations have a differential effect on Na+,K+-ATPase activity and ouabain binding

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na⁺,K⁺-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na⁺,K⁺-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K⁺/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na⁺,K⁺-ATPase. Functional impairment of Na⁺,K⁺-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.     

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na⁺, Cl⁻, and K⁺ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na⁺-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca²⁺ (but not other cations) to functionally replace Na⁺ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca²⁺ and Na⁺ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca²⁺ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na⁺-binding sites.     

The Second Sodium Site in the Dopamine Transporter Controls Cation Permeation and Is Regulated by Chloride

The dopamine transporter (DAT) belongs to the family of neurotransmitter:sodium symporters and controls dopamine (DA) homeostasis by mediating Na⁺- and Cl⁻-dependent reuptake of DA. Here we used two-electrode voltage clamp measurements in Xenopus oocytes together with targeted mutagenesis to investigate the mechanistic relationship between DAT ion binding sites and transporter conductances. In Li⁺, DAT displayed a cocaine-sensitive cation leak current ∼10-fold larger than the substrate-induced current in Na⁺. Mutation of Na⁺ coordinating residues in the first (Na1) and second (Na2) binding sites suggested that the Li⁺ leak depends on Li⁺ interaction with Na2 rather than Na1. DA caused a marked inhibition of the Li⁺ leak, consistent with the ability of the substrate to interact with the Li⁺-occupied state of the transporter. The leak current in Li⁺ was also potently inhibited by low millimolar concentrations of Na⁺, which according to our mutational data conceivably depended on high affinity binding to Na1. The Li⁺ leak was further regulated by Cl⁻ that most likely increases Li⁺ permeation by allosterically lowering Na2 affinity. Interestingly, mutational lowering of Na2 affinity by substituting Asp-420 with asparagine dramatically increased cation permeability in Na⁺ to a level higher than seen in Li⁺. In addition to reveal a functional link between the bound Cl⁻ and the cation bound in the Na2 site, the data support a key role of Na2 in determining cation permeability of the transporter and thereby possibly in regulating the opening probability of the inner gate.     

An unusual conformation from Na+-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na+-binding

The Na⁺,K⁺-ATPase (NKA) and non-gastric H⁺,K⁺- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing (E2) conformations and differ in their exported substrate (Na⁺ or H⁺) and stoichiometries (3 Na⁺:2 K⁺ or 1 H⁺:1 K⁺). We reported that structures of the NKA-mimetic ngHKA mutant K794S/A797P/W940/R949C (SPWC) with 2 K⁺ occluded in E2-P_i and 3 Na⁺-bound in E1·ATP states were nearly identical to NKA structures in equivalent states. Here we report the cryo-EM structures of K794A and K794S, two poorly-selective ngHKA mutants, under conditions to stabilize the E1·ATP state. Unexpectedly, the structures show a hybrid with both E1- and E2-like structural features. While transmembrane segments TM1-TM3 and TM4's extracellular half adopted an E2-like conformation, the rest of the protein assumed an E1 configuration. Two spherical densities, likely bound Na⁺, were observed at cation-binding sites I and III, without density at site II. This explains the E2-like conformation of TM4's exoplasmic half. In NKA, oxygen atoms derived from the unwound portion of TM4 coordinated Na⁺ at site II. Thus, the lack of Na⁺ at site II of K794A/S prevents the luminal portion of TM4 from taking an E1-like position. The K794A structure also suggests that incomplete coordination of Na⁺ at site III induces the halfway rotation of TM6, which impairs Na⁺-binding at the site II. Thus, our observations provide insight into the molecular mechanism of E2-E1 transition and cooperative Na⁺-binding in the NKA and other related cation pumps.     

Intracellular Proton-Transfer Mutants in a CLC Cl?/H+ Exchanger

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl⁻ and H⁺. Mutational analysis based on the known structure reveals several key residues required for coupling H⁺ to the stoichiometric countermovement of Cl⁻. E148 (Glu_ex) transfers protons between extracellular water and the protein interior, and E203 (Glu_in) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H⁺ transport while preserving Cl⁻ transport. We tested the role of Glu_in by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H⁺/Cl⁻ exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H⁺ coupling and severely inhibit Cl⁻ flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H⁺ transport with substantial Cl⁻ transport. Influence on H⁺ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Glu_in is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl⁻ movements to bring about Cl⁻/H⁺ exchange.     

Identification of a 3rd Na+ Binding Site of the Glycine Transporter,GlyT2

The Na⁺/Cl^- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na⁺ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na⁺ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na⁺-dependent leucine transporter, LeuT_Aa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na⁺ binding sites in GlyT2, but the physical location of the third Na⁺ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuT_Aa. Although betaine transport by BetP is coupled to the co-transport of two Na⁺ ions, the first Na⁺ site is not conserved between BetP and LeuT_Aa, the so called Na1'' site. We hypothesized that the third Na⁺ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1'' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na⁺ placed at the location consistent with the Na1'' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na⁺ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.     

The ascidian sperm reaction: evidence for Cl- and HCO3- involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na⁺ concentration of the medium. The optimal [Na⁺] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K⁺] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K⁺ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl^?-free (NO^?₃ or glutamate substituted) seawater (SW). Cl^? efflux accompanies H⁺ efflux with twice as many Cl^? being released as H⁺. Both H⁺ and Cl^? release in Cl^?-free SW are dependent upon CO₂ being present in HCO^?₃-free medium, suggesting that H⁺ efflux is in part Cl^? and HCO^?₃-mediated. However, the chloride channel blocking agent SITS has no effect on H⁺ release and augments Cl^? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na⁺-dependent acidification system of unreacted sperm and the Cl^?- and HCO^?₃-mediated H⁺ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.     

Na+ transport processes in isolated guinea pig nasal gland acinar cells

In the dispersed acinar cells of the submucosal nasal gland in the guinea pig, intracellular Na⁺ concentration ([Na⁺]i) was measured with a microfluorimetric imaging method and the cytosolic indicator dye, sodium-binding benzofuran isophthalate, under HCO₃^?-free conditions. In the unstimulated condition, the [Na⁺]i was averaged to 12.8 ± 5.2 mM. Addition of 100 μM ouabain or removal of external K⁺ caused an increase in [Na⁺]i. Replacement of external Cl^? with NO₃^? or addition of 0.5 mM furosemide reversibly decreased the [Na⁺]i. The recovery process from the reduced [Na⁺]i was inhibited by removal of either K⁺ or Cl^? in the bath solution. These findings indicate the presence of a continuous influx of Na⁺ coupled with K⁺ and Cl^? movement. Application of acetylcholine (ACh, 1 μM) caused an increase in [Na⁺]i by about 15–20 mM, which was completely inhibited by addition of 10 μM atropine. Increased cytosolic Na⁺ induced by ACh was extruded by the Na⁺-K⁺ pump. Removal of external Cl^? and addition of 50 μM dimethylamiloride inhibited ACh-induced increase in [Na⁺]i by about 66% and 19%, respectively. In both unstimulated and stimulated state, Na⁺-K⁺ pump, Na-K-Cl cotransport, and Na⁺-H⁺ exchange play a critical role in maintaining intracellular electrolyte environment and in controlling a continuous secretion of nasal fluids. © 1995 Wiley-Liss, Inc.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

cAMP activates Cl−/HCO3− exchange for regulation of intracellular pH in renal epithelial cells

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK₁ cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na⁺-independent Cl^?/HCO₃^? exchange, and by removal of extracellular Cl^?. In addition, Bt₂ cAMP causes Cl^? entry into LLC-PK₁ cells. These results suggest that cAMP activates Cl^? transport, namely Na⁺-independent Cl^?/HCO₃^? exchange, which participates in pH_i regulation.     

Molecular Characterization and In Situ Localization of a Mouse Retinal Taurine Transporter

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na⁺- and Cl^?-dependent [³H]taurine transport activity in Xenopus laevis oocytes that saturated with an average K_m of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl^? was varied, a single saturation process with an average apparent equilibrium constant (K_Cl?) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na⁺ was varied (apparent equilibrium constant, K_Na+～54.8 mM). Analyses of the Na⁺- and Cl^?-concentration dependence data suggest that at least two Na⁺ and one Cl^? are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β-alanine > l -diaminopropionic acid > guanidinoethane sulfonate > β-guanidinopropionic acid > chloroquine > γ-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.     

Modulation of Na<Superscript>+</Superscript>/Mg<Superscript>2+</Superscript> exchanger stoichiometry ratio by Cl<Superscript>−</Superscript> ions in basolateral rat liver plasma membrane vesicles

The Na⁺/Mg²⁺ exchanger represents the main Mg²⁺ extrusion mechanism operating in mammalian cells including hepatocytes. We have previously reported that this exchanger, located in the basolateral domain of the hepatocyte, promotes the extrusion of intravesicular trapped Mg²⁺ for extravesicular Na⁺ with ratio 1. This electrogenic exchange is supported by the accumulation of tetraphenyl-phosphonium within the vesicles at the time when Mg²⁺ efflux occurs. In this present study, the role of extra- and intra-vesicular Cl^? on the Na⁺/Mg²⁺ exchange ratio was investigated. The results reported here suggest that Cl^? ions are not required for the Na⁺ to Mg²⁺ exchange to occur, but the stoichiometry ratio of the exchanger switches from electrogenic (1Na _in ⁺ :1 Mg _out ²⁺ ) in the presence of intravesicular Cl^? to electroneutral (2Na _in ⁺ :1 Mg _out ²⁺ ) in their absence. In basolateral liver plasma membrane vesicles loaded with MgCl₂ labeled with ³⁶Cl^?, a small but significant Cl^? efflux (~30 nmol Cl^?/mg protein/1 min) is observed following addition of NaCl or Na-isethionate to the extravesicular medium. Both Cl^? and Mg²⁺ effluxes are inhibited by imipramine but not by amiloride, DIDS, niflumic acid, bumetanide, or furosemide. In vesicles loaded with Mg-gluconate and stimulated by Na-isethionate, an electroneutral Mg²⁺ extrusion is observed. Taken together, these results suggest that the Na⁺/Mg²⁺ exchanger can operate irrespective of the absence or the presence of Cl^? in the extracellular or intracellular environment. Changes in trans-cellular Cl^? content, however, can affect the modus operandi of the Na⁺/Mg²⁺ exchanger, and consequently impact "cellular" Na⁺ and Mg²⁺ homeostasis as well as the hepatocyte membrane potential.     

Dynamic lipid interactions in the plasma membrane Na+,K+-ATPase

The function of ion-transporting Na⁺,K⁺-ATPases depends on the surrounding lipid environment in biological membranes. Two established lipid-interaction sites A and B within the transmembrane domain have been observed to induce protein activation and stabilization, respectively. In addition, lipid-mediated inhibition has been assigned to a site C, but with the exact location not experimentally confirmed. Also, possible effects on lipid interactions by disease mutants dwelling in the membrane-protein interface remain relatively uncharacterized. We simulated human Na⁺,K⁺-ATPase α₁β₁FXYD homology models in E1 and E2 states in an asymmetric, multicomponent plasma membrane to determine both wild-type and disease mutant lipid-protein interactions. The simulated wild-type lipid interactions at the established sites A and B were in agreement with experimental results thereby confirming the membrane-protein model system. The less well-characterized, proposed inhibitory site C was dominated by lipids lacking inhibitory properties. Instead, two sites hosting inhibitory lipids were identified at the extracellular side and also a cytoplasmic CHL-binding site that provide putative alternative locations of Na⁺,K⁺-ATPase inhibition. Three disease mutations, Leu302Arg, Glu840Arg and Met859Arg resided in the lipid-protein interface and caused drastic changes in the lipid interactions. The simulation results show that lipid interactions to the human Na⁺,K⁺-ATPase α₁β₁FXYD protein in the plasma membrane are highly state-dependent and can be disturbed by disease mutations located in the lipid interface, which can open up for new venues to understand genetic disorders.     

The ionic relations of Porphyra purpurea(Roth) C.Ag. (Rhodophyta, Bangiales)

Abstract Radioisotope equilibration techniques have been used to determine the intracellular concentration of K⁺, Na⁺ and Cl_?, together with the unidirectional ion fluxes across the plasmalemma of Porphyra purpurea. Influx and efflux of ⁴²K⁺, ²⁴Na⁺ and ³⁶C1^? are biphasic, the rapid, initial uptake and loss of tracer from individual thalli being attributable to desorption from extracellular regions. Cellular fluxes are slower and monophasic, cells discriminating in favour of K⁺ and Cl^? and against Na⁺. A comparison between the equilibrium potential of individual ion species and the measured membrane potential demonstrates that there is an active component of K⁺ and Cl^? influx and Na⁺ efflux. ‘Active’ uptake and ‘passive’ loss of K⁺ and Cl^? are reduced when plants are kept in darkness, suggesting that a fraction of the transport of K⁺ and Cl^? may be due to ‘exchange diffusion’ (K⁺/K⁺ and Cl^?/Cl^?antiport).     

A Multisubstrate Kinetic Mechanism of Dopamine Transport in the Nucleus Accumbens and Its Inhibition by Cocaine

Abstract: Kinetic studies of dopamine transport into suspensions of nucleus accumbens (NAcc) and effects of Na⁺ and Cl^? as cosubstrates were performed using rotating disk electrode voltammetry. To mimic chemical neurotransmission, dopamine was added as a rapid pulse, and transporter-mediated clearance of dopamine was evaluated kinetically. This paradigm was shown to approximate a zero trans entry transport experiment. Dopamine was taken up with apparent K_m and V_max values of 1.3 µM and 375 pmol/s/g wet weight, respectively. Transport exhibited apparent trans acceleration. Substitution of Na⁺ with choline or Cl^? with isethionate reduced dopamine transport with reaction orders of two and unity, respectively, accompanied by reductions in V_max with no changes in K_m. Apparent K_Na and K_Cl values were 70.0 and 92.1 mM, respectively. Dopamine transport in NAcc was found to follow a partially random, sequential mechanism in which dopamine and Na⁺ bind randomly to the transporter followed by binding of Cl^? before transport. Cocaine inhibited dopamine transport and the influences of the other substrates allosterically with an overall K_i of 0.30 µM. Thus, the general kinetic mechanism of the transport of dopamine in the NAcc is identical to that previously reported by this laboratory for dopamine transport in the striatum. However, the dopamine transporter in the NAcc is more tightly regulated by Na⁺, possesses a higher kinetic turnover rate, is four times more sensitive to cocaine than the striatal transporter, and exhibits cocaine inhibition independent of [substrate]. These findings suggest that cocaine modulates chemical signaling in NAcc differently than in striatum, providing down-regulation of function irrespective of [substrate], thereby enhancing dopaminergic signaling more robustly in the NAcc than in the striatum.     

Fluxes and compartmentation of K+, Na+ and Cl−, and action of auxins in suspension-cultured Petroselinum cells

Transport of ⁸⁶Rb⁺/K⁺, ²²Na⁺, ³⁶Cl^?, and [³H]indole acetic acid (IAA) has been studied on suspension-cultured cells of the parsley, Petroselinum crispum (Mill) Nym. By compartmental analysis two intracellular compartments of K⁺, Na⁺, and Cl^? have been identified and ascribed to the cytoplasm and vacuole; half-times of exchange were around 200 s and 5 h, respectively. According to the Ussing-Teorell flux equation, active transport is required for the influx into the cytoplasm at the plasmalemma (K⁺, Cl^?) and the tonoplast (K⁺, Na⁺, Cl^?). The plasmalemma permeability pattern, P_K:P_Na:P_Cl=1.00:0.24:0.38, features an increased chloride permeability compared with cells from higher plant tissues. IAA uptake showed an exponential timecourse, was half-maximal after 10 min, and a linear function of the IAA concentration from 10^?9 to 10^?5 M. IAA and 2,4-dichlorophenoxy acetic acid reduce the apparent influx of K⁺, Na⁺, Cl^? during the initial 30 min after addition and subsequently accelerate both in- and efflux of these ions. We discuss that auxins could affect the ion fluxes in a complex way, e.g. by protonophorous activity and by control of the hypothetical proton pump.     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.